Nimotuzumab Site-Specifically Labeled with 89Zr and 225Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers

Abstract

To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH2) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH2 or SH groups can require extreme conditions and may affect target recognition/binding and must therefore be tested. In the present study, nimotuzumab was site-specifically labeled using ∆N-SpyCatcher/SpyTag with different chelators and radiometals. Nimotuzumab is a well-tolerated anti-EGFR antibody with low skin toxicities. First, ΔN-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive ΔN-SpyCatcher-DFO. The ΔN-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag-∆N-SpyCatcher-DFO. Radiolabeling was performed with 89Zr, and the conjugate was used for the in vivo microPET imaging of EGFR-positive MDA-MB-468 xenografts. Similarly, ∆N-SpyCatcher was conjugated to an eighteen-membered macrocyclic chelator macropa-maleimide and used to radiolabel nimotuzumab-SpyTag with actinium-225 (225Ac) for in vivo radiotherapy studies. All constructs were characterized using biolayer interferometry, flow cytometry, radioligand binding assays, HPLC, and bioanalyzer. MicroPET/CT imaging showed a good tumor uptake of 89Zr-nimotuzumab-SpyTag-∆N-SpyCatcher with 6.0 ± 0.6%IA/cc (n = 3) at 48 h post injection. The EC50 of 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher and 225Ac-control-IgG-SpyTag-∆N-SpyCatcher against an EGFR-positive cell-line (MDA-MB-468) was 3.7 ± 3.3 Bq/mL (0.04 ± 0.03 nM) and 18.5 ± 4.4 Bq/mL (0.2 ± 0.04 nM), respectively. In mice bearing MDA-MB-468 EGFR-positive xenografts, 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher significantly (p = 0.0017) prolonged the survival of mice (64 days) compared to 225Ac-control IgG (28.5 days), nimotuzumab (28.5 days), or PBS-treated mice (30 days). The results showed that the conjugation and labeling using SpyTag/∆N-SpyCatcher to nimotuzumab did not significantly (p > 0.05) alter the receptor binding of nimotuzumab compared with a non-specific conjugation approach. 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model.

SPECT imaging of 226Ac as a theranostic isotope for 225Ac radiopharmaceutical development

“…Currently, 225 Ac radiopharmaceuticals are typically paired with alternative imageable isotopes including 68 Ga, 177 Lu, 111 In, and 89 Zr (Baum and Kulkarni 2012, Kratochwil et al 2016, Kelly et al 2020, Solomon et al 2020. 177 Lu has been used for intra-therapy dosimetry of 225 Ac as it is often administered in tandem therapies (Khreish et al 2020, Rosar et al 2021.…”

“…Due to a lack of an element-equivalent imageable isotope of 225 Ac, performance evaluation of 225 Ac radiopharmaceuticals is currently assessed by either performing invasive organ-harvesting biodistribution studies in preclinical models. In clinical settings, 225 Ac radiopharmacuticals are paired with an imageable isotope, such as 68 Ga (Baum and Kulkarni 2012), 177 Lu (Kratochwil et al 2016), 111 In (Kelly et al 2020) and 89 Zr (Solomon et al 2020). Absorbed dose is inferred with the underlying assumption of equivalent uptake and residency between 225 Ac and imageable forms of a radiopharmaceutical using other isotopes.…”

[225Ac]Ac/[89Zr]Zr-labeled N4MU01 radioimmunoconjugates as theranostics against nectin-4 positive triple negative breast cancer

“…N4MU01 was conjugated with p -SCN-Bn-deferoxamine (DFO) for labeling with 89 Zr as described previously [22]. Quality control of the immunoconjugate was done using size exclusion chromatography (SEC) high-performance liquid chromatography (HPLC) (SEC-HPLC), flow cytometry, and automated electrophoresis (2100 Bioanalyzer, Agilent, Santa Clara, CA, USA).…”

“…Quality control of the immunoconjugate was done using SEC-HPLC, matrix-assisted laser desorption (MALDI) time of flight (TOF) (MALDI-TOF) and flow cytometry. Radiolabeling with 225 Ac was performed as previously reported [22].…”

PET and SPECT Imaging of the EGFR Family (RTK Class I) in Oncology

“…The calculated absorbed dose of [ 89 Zr]Zr-nimotuzumab was lower than what was earlier estimated for [ 89 Zr]Zr-DFO-panitumumab [ 121 ], suggesting that [ 89 Zr]Zr-nimotuzumab might be a promising alternative to panitumumab and also cetuximab [ 123 ]. A recent study also reported 89 Zr labeling of nimotuzumab with a SpyTag/SpyCatcher, which would also enable labeling with 225 Ac for radioimmunotherapy [ 124 ]. Currently, [ 89 Zr]Zr-nimotuzumab is investigated in a phase I/II trial to determine its diagnostic quality in lung and colorectal cancer patients (NCT04235114).…”